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1 Article 2 Outer membrane protein A conservation among 3 tsutsugamushi isolates suggests its potential 4 as a protective and diagnostic target

5 Sean M. Evans1,†,‡, Haley E. Adcox1‡, Lauren VieBrock1, Ryan S. Green1, Alison Luce-Fedrow2,3, 6 Suschsmita Chattopadhyay2, Ju Jiang2, Richard T. Marconi1, Daniel Paris4, Allen L. Richards2,5, 7 and Jason A. Carlyon1*

8

9 1 Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, 10 School of Medicine, Richmond, Virginia, 23298, USA; [email protected]; [email protected]; 11 [email protected]; [email protected]; [email protected]; 12 [email protected] 13 2 Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, 14 20910, USA; [email protected]; [email protected]; 15 [email protected] 16 3 Department of Biology, Shippensburg University, Shippensburg, Pennsylvania, 17257, USA; 17 [email protected] 18 4 Department of Medicine, Swiss Tropical and Public Health Institute, Basel, Switzerland; 19 [email protected] 20 5 Department of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, 21 Bethesda, Maryland, 20814, USA 22 23 24 * Correspondence: [email protected]; Tel.: +1-804-628-3382 25 † Present address: Virology and Serology Department, Infectious Diseases Laboratory, Utah Public Health 26 Lab, Taylorsville, UT 84129, USA 27 ‡ These authors contributed equally to this work. 28 29

30 Abstract: Scrub threatens one billion people in the Asia-Pacific area and cases have emerged 31 outside this region. It is caused by infection with any of the multitude of strains of the bacterium, 32 . A vaccine that affords heterologous protection and a commercially available 33 molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated 34 amino acid sequences of outer membrane protein A (OmpA) are highly conserved among 51 O. 35 tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. 36 tsutsugamushi OmpA to be very similar to that of the phylogenetically related pathogen, Anaplasma 37 phagocytophilum, including the location of a helix that contains residues functionally essential for A. 38 phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. 39 tsutsugamushi strains, but not from negative control . Using these primers in quantitative 40 real-time PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from 41 organs of mice that had been experimentally infected with the Karp or Gilliam strains. The high 42 degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a 43 molecular diagnostic target and justifies its consideration as a candidate for developing a broadly 44 protective vaccine.

45 Keywords: Scrub typhus; Orientia tsutsugamushi; ; ; outer membrane protein A; 46 Anaplasma 47

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48 1. Introduction

49 Scrub typhus is an acute, febrile, and potentially deadly disease caused by infection with the 50 larval -vectored bacterium, Orientia tsutsugamushi. Long known to be endemic 51 to the Asia-Pacific, a densely-populated region where more than one million cases are estimated to 52 occur annually, scrub typhus affects all organs and the central nervous system. Clinical 53 manifestations can include , , , maculopapular rash, , 54 pneumonitis, , and coagulopathies that can result in circulatory system collapse (reviewed 55 in [1,2]). The disease can account for up to 20% of all acute undifferentiated febrile episodes and up 56 to 27% of blood culture-negative fever patients in endemic areas [3-5]. Its non-specific clinical 57 presentation makes clinical diagnosis very difficult [6]. Mortality rates range from less than 1.4% to 58 approximately 70%, depending on prior patient immune status, whether proper antibiotic treatment 59 is initiated in a timely manner, and the bacterial strain causing the infection [1,7-9]. Recent outbreaks 60 in the Asia-Pacific [10-23], as well evidence for scrub typhus or scrub typhus-like infections in 61 Cameroon, Kenya, Congo, Djibouti, Tanzania, Chile, and Peru signify these illnesses as both 62 emerging and reemerging global diseases of global importance [24-30].

63 The genus Orientia is a member of the order Rickettsiales, which contains other arthropod vector- 64 transmitted pathogens, including Anaplasma, Ehrlichia, and Rickettsia. Until recently, the genus 65 consisted of a single species, Orientia tsutsugamushi, which contained multiple antigenically distinct 66 strains [31]. In 2010, a second agent, Candidatus Orientia chuto, was discovered in a febrile patient 67 presenting with a scrub typhus-like illness that had been acquired in the United Arab Emirates [32]. 68 The extensive immunogenic diversity among O. tsutsugamushi strains has contributed to the inability 69 to develop a scrub typhus vaccine that achieves heterologous protection despite more than seven 70 decades’ worth of efforts [6]. No commercially available molecular diagnostic assay for the disease 71 exists. Serology-based tests suffer from a high seroprevalence baseline among populations living in 72 scrub typhus endemic areas. While polymerase chain reaction (PCR)-based tests can overcome 73 limitations of serologic assays, only a limited number of Orientia spp. nucleic acid sequences have 74 been explored for their potential as molecular diagnostic targets [33-37].

75 Outer membrane protein A (OmpA; also referred to as -associated lipoprotein) is 76 conserved among most Gram-negative bacteria and contributes to the virulence of Gram-negative 77 pathogens, especially their abilities to adhere to and invade host cells [38-45]. Antisera raised against 78 entire OmpA proteins or specific binding domains thereof for Anaplasma spp., E. chaffeensis, and R. 79 conorii inhibit bacterial invasion of host cells in vitro [38,41,42,44,45]. These Rickettsiales members 80 express OmpA during infection of human patients and/or experimentally infected animals [38,44,46]. 81 Several Rickettsiales species and strains have stretches of ompA DNA sequences that exhibit high 82 degrees of identity [44,45,47,48], which suggests their potential as effective nucleic acid-based 83 diagnostic targets. Limited evidence suggests that OmpA antibodies offer at least some protection 84 from rickettsial infections in vivo [49]. While O. tsutsugamushi Ikeda expresses OmpA during infection 85 of mammalian host cells in vitro [50], ompA conservation among Orientia spp. and whether these 86 bacteria express ompA during in vivo infection has yet to be examined.

87 In this study, we determined that ompA DNA and translated amino acid sequences are highly 88 conserved among 51 geographically diverse O. tsutsugamushi isolates. Molecular modeling revealed 89 the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of Anaplasma 90 phagocytophilum OmpA, including the location of a helix and residues thereof that are essential for 91 Anaplasma spp. OmpA function. A PCR primer pair was developed that amplified ompA DNA from 92 all O. tsutsugamushi strains examined and enabled sensitive detection and quantitation of O. 93 tsutsugamushi ompA DNA from organs of experimentally infected mice. The high degree of 94 conservation of OmpA among O. tsutsugamushi isolates suggests that it be considered both as a 95 diagnostic target and potential antigen for developing a broadly protective scrub typhus vaccine.

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96 2. Materials and Methods

97 2.1 Orientia spp. DNA samples examined in this study

98 Nearly all of the O. tsutsugamushi strains examined in this study have been previously described 99 [32,51-64]. The isolates, their countries of origin, publication in which they were originally reported, 100 and their ompA GenBank accession numbers and locus tags are listed in Table 1.

101 Table 1. Orientia spp. isolates used in this study

Isolate Geographic Origin Reference ompA GenBank Accession Number or Locus Tag

18-032460 Malaysia 63 MH167971

AFC3 Thailand 56 MH167972

AFC30 Thailand NRa MH167973

AFPL12 Thailand NR MH167974

Boryong South Korea 54 OTBS_RS08365

Brown Australia 61 MH167975

Calcutta India NR MH167976

Citrano Australia 61 MH167977

CRF09 Northern Thailand 52 MH167978

CRF58 Northern Thailand 52 MH167979

CRF136 Northern Thailand 52 MH167980

CRF158 Northern Thailand 52 MH167981

FPW1038 Western Thailand 53 MH167982

FPW2016 Western Thailand 53 MH167983

FPW2049 Western Thailand 53 MH167984

Gilliam Burma 57 MH167985

Ikeda Japan 58 OTT_RS06375

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Karp New Guinea 55 OTSKARP_0358

Kato Japan 64 OTSKATO_0610

LNT1153 Northwestern Laos 51 MH167986

LNT1189 Northwestern Laos 51 MH167987

LNT1301 Northwestern Laos 51 MH167988

LNT1310 Northwestern Laos 51 MH167989

MAK110 Taiwan 62 MH167990

MAK119 Taiwan 62 MH167991

MAK243 Taiwan 62 MH167992

SV400 Southern Laos 51 MH167993

SV445 Southern Laos 51 MH167994

SV484 Southern Laos 51 MH167995

TA763 Thailand 59 OTSTA763_0977

TH1812 Thailand 59 MH167996

TH1817 Thailand 59 MH167997

TM2328 Central Laos 51 MH167998

TM2395 Central Laos 51 MH167999

TM2494 Central Laos 51 MH168000

TM2532 Central Laos 51 MH168001

TM2766 Central Laos 51 MH168002

TM2950 Central Laos 51 MH168003

TM3115 Central Laos 51 MH168004

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UT76 Northeastern Thailand 53 MH168005

UT125 Northeastern Thailand 53 MH168006

UT169 Northeastern Thailand 53 MH168007

UT177 Northeastern Thailand 53 MH168008

UT219 Northeastern Thailand 53 MH168009

UT336 Northeastern Thailand 53 MH168010

UT340 Northeastern Thailand 53 MH168011

UT418 Northeastern Thailand 53 MH168012

UT559 Northeastern Thailand 53 MH168013

UT652 Northeastern Thailand 53 MH168014

Volner New Guinea 60 MH168015

Wood Australia 61 MH168016

102 aNR, no previous published report.

103

104 2.2 PCR, cloning, and DNA sequence analyses

105 PCR was performed using isolated O. tsutsugamushi strain DNA and MyTaq polymerase Red 106 (Bioline, Taunton, MA) following the manufacturer’s instructions. Following an initial denaturing 107 step at 95°C for 1 min, thermal cycling conditions were 35 cycles of 95°C for 15 s, 55°C for 15 s, and 108 72°C for 10 s, followed by a final extension at 72°C for 20 s. Amplicons were analyzed in 2.0% agarose 109 gels in 40 mM tris-acetate-2 mM EDTA (pH 8.5). Primer sequences targeting ompA were designed 110 according to ompA (OTT_RS06375) of the annotated O. tsutsugamushi Ikeda genome [65] and are listed 111 in Table 2. DNA samples that yielded amplicons of the expected sizes were again subjected to PCR 112 using the appropriate primer sets and Platinum HiFi Taq polymerase (Thermo Fisher, Waltham, MA) 113 according to the manufacturer’s instructions. Platinum HiFi Taq polymerase thermal cycling 114 conditions consisted of an initial denaturation step of 94°C for 2 min, followed by 34 cycles of 94°C 115 for 15 s, 55°C for 30 s, and a final extension step at 68°C for 1 min. The resulting amplicons were 116 subjected to agarose gel electrophoresis, after which they were visualized using a Blue View 117 Transilluminator (Vernier Biotechnology, Beaverton, OR), excised, and purified using the 118 QIAquickGel Extraction Kit (Qiagen, Valencia, CA). The purified PCR products were TA-cloned into 119 pCR2.1-TOPO using the TOPO TA Cloning kit (Thermo Fisher). Clones were transformed into 120 chemically competent TOPO and incubated for 1 h in SOC medium (Thermo Fisher) 121 at 37°C with agitation at 250 RPM. Aliquots of each culture were plated onto Luria-Bertani (LB) agar 122 plates containing 0.1 mg/mL ampicillin and incubated at 37°C overnight. Colony PCR using vector-

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123 derived M13F and M13R primers was performed to identify colonies that harbored plasmids 124 containing inserts of the expected size. Plasmids isolated from PCR-positive colonies using the 125 PerfectPrep Spin Mini Kit (5 Prime, Gaithersburg, MD) were sequenced using M13F and M13R 126 primers by Genewiz (South Plainfield, NJ) and the provided sequences were analyzed using the 127 Lasergene 7.1 software package (DNASTAR, Madison, WI).

128 Table 2. Oligonucleotide primers used in this study

Primer Designationa Sequence (5’ to 3’)

OTT_RS06375-1F ATGATTAAAAAGTCAATTATTAGTATATGTGTATTAGTGC

OTT_RS06375-615R CTATGCTATATTACTTTTAATAATTGTGACAGACC

OTT_RS06375-64F TGTTTATGGCAAAGATCTAAACATAGTAAC

ompA-57F GTGGAAATGTTTATGGCAAAGATCTAAAC

ompA-260R GCTTGTAAAAACTGTTCATGCTTTACATC

Eubacterial 16S-F GTTCGGAATTACTGGGCGTA

Eubacterial 16S-R AATTAAACCGCATGCTCCAC

R17K-135 ATGAATAAACAACGKbCANGGHACAC

R17K-249 RAAGTAATGCRCCTACACCTACTC

129 aF and R refer to primers that bind to the sense and antisense strand, respectively. The number immediately 130 preceding the F or R denotes the first nucleotide position where the primer binds.

131 bDegenerate positions contained equal molar base concentrations of the following nucleotides: (K), guanine and 132 thymine, (N), adenine, guanine, thymine, and cytosine; (H), adenine, guanine, and thymine; (R) adenine and 133 guanine.

134 2.3 In silico analyses and GenBank accession numbers of O. tsutsugamushi strain ompA sequences

135 O. tsutsugamushi strain ompA sequences were aligned using MegAlign and translated using 136 EditSeq, both of which are part of the Lasergene 7.1 software package (DNASTAR). New O. 137 tsutsugamushi strain ompA sequences identified herein have been deposited in GenBank with 138 accession numbers listed in Table 1. Sequence distances and percent similarity of O. tsutsugamushi 139 OmpA proteins were calculated using the ClustalW option in MegAlign. Heat maps indicating 140 similarity/diversity of the O. tsutsugamushi strain OmpA nucleotide and translated protein sequences 141 were generated using HEATMAP hierarchical clustering web tool 142 (www.hiv.lanl.gov/content/sequence/HEATMAP/heatmap.html/). To predict a putative tertiary 143 structure for O. tsutsugamushi Ikeda OmpA, the mature (minus the signal sequence) Ikeda sequence 144 (residues 21 to 204) was threaded onto solved crystal structures of proteins with similar sequences 145 using the PHYRE2 (Protein Homology/analogy Recognition Engine, version 2.0) server 146 (www.sbg.bio.ic.ac.uk/phyre2) [66]. Six templates (c4zhvB [bacterial signaling protein Yfib], c5jirB

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147 [oop family OmpA-OmpF porin; Treponema pallidum protein tp0624], c3s0yA [periplasmic domain of 148 motility protein B (MotB) residues 64-256], c2l26A [uncharacterized Mycobacterium tuberculosis 149 membrane protein rv0899/mt0922], c2k1sA [folded C-terminal fragment of YiaD from E. coli], and 150 c5wtlB [periplasmic portion of outer membrane protein 2a (OmpA) from Capnocytophaga gingivalis]) 151 were selected to model OmpA based on heuristics to maximize confidence, percentage amino acid 152 identity, and alignment coverage. For the final model, 92% of the protein was modelled at greater 153 than 90% confidence. Residues that vary among the OmpA translated sequences identified in this 154 study were denoted on the O. tsutsugamushi Ikeda OmpA PHYRE2 model using the PyMOL 155 algorithm (pymol.org/educational).

156 2.4 Confirmation of ompA PCR primer specificity

157 Based on an alignment of all ompA sequences determined herein, primers ompA-57F and ompA- 158 260R (Table 2) were designed to amplify nucleotides 57 to 260 based on O. tsutsugamushi Ikeda ompA 159 (OTT_RS06375) [65]. To confirm that the primers were specific for O. tsutsugamushi ompA, they were 160 utilized in PCR reactions that included genomic DNA from numerous Rickettsia species (R. africae, R. 161 akari, R. australis, R. conorii, R. montanensis, R. parkeri, R. rhipicephali, R. rickettsii, R. sibirica, R. prowazekii, 162 R. typhi, and R. amblyommii), as well as other bacterial species (, E. coli, Legionella 163 pneumophila, vinsonii, risticii, N. sennetsu, and Francisella persica) using thermal 164 cycling conditions and agarose gel electrophoresis described above. To verify that the control 165 templates and thermal cycling conditions were amenable for PCR amplification, reactions were 166 simultaneously performed on the Rickettsia species using degenerate primers that targeted the 167 Rickettsia 17-kDa gene, R17K-135F and R17K-249R [36], and on the other bacterial species using 168 primers that targeted a conserved eubacterial 16S rRNA sequence (Table 2) [36].

169 2.5 Mice

170 Female six- to eight-week old CD-1 Swiss outbred mice (Charles River Laboratories, Wilmington, 171 MA) were housed in animal (ABSL)-2 laboratories prior to inoculation. Two days prior 172 to inoculation, they were relocated to an ABSL-3 laboratory to adapt to their new surroundings. The 173 mice were intradermally inoculated with 103 MuID50 of O. tsutsugamushi Karp or Gilliam strains 174 produced from liver-spleen homogenate of infected CD-1 mice into the dorsum of the right ear as 175 previously described [67]. Sterile PBS was used as mock inoculum to inject negative control animals 176 [67]. At various days post-infection, the mice were euthanized, and organs harvested for DNA 177 isolation [68]. All animal research was performed under the approval of the Institutional Animal Care 178 and Use Committee at the Naval Medical Research Center (Protocol Number: 11-IDD-26.

179 2.6 Quantitative real-time PCR (qPCR)

180 To generate an ompA DNA standard, ompA nucleotides 1 to 615 were amplified using the ompA- 181 1F/615R primer set and Platinum HiFi Taq polymerase. The amplicon was gel-purified and cloned 182 into pCR2.1-TOPO as described above. Concentration (in ng/l) of the resulting plasmid, pCR2.1- 183 ompA, was determined by UV spectrophotometry. The concentration was converted to copies/l 184 using the EndMemo DNA/RNA Copy Number Calculator 185 (http://endmemo.com/bio/dnacopynum.php). To evaluate the sensitivity of the ompA-57F/260R 186 primer set, triplicate 20 l reactions containing either dilutions of pCR2.1-ompA ranging from 1 x 106 187 to 1 x 10-2 copies/l or no template were subjected to qPCR using SsoFast EvaGreen Supermix (Bio- 188 Rad, Hercules, CA) in a CFX96 Real-Time System thermocycler (Bio-Rad). Thermal cycling conditions 189 consisted of an initial denaturation step of 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 190 55°C for 30 s. The ability of the ompA-57F/260R primer set to detect ompA in DNA isolated from mouse 191 tissues recovered from O. tsutsugamushi infected mice was also assessed. Using the DNeasy Blood 192 and Tissue kit (Qiagen), DNA was isolated from the heart, kidney, liver, lung, and spleen harvested 193 on various days post infection from Swiss CD-1 mice that had been intradermally inoculated with

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194 either the O. tsutsugamushi Gilliam or Karp strain or uninfected control mice [68]. Fifty ng of mouse 195 tissue DNA template per sample was subjected to qPCR exactly as described for the ompA DNA 196 standards. Infrequently, an individual ompA-57/260R reaction using uninfected mouse tissue DNA 197 as template or no template control would generate a Cq value at cycle 36.5 or later. In such 198 experiments, only earlier Cq values generated for infected samples were considered as specifically 199 amplifying ompA.

200 3. Results 201 3.1 Analyses of O. tsutsugamushi ompA DNA and translated amino acid sequences 202 203 DNA samples recovered from 51 geographically diverse O. tsutsugamushi isolates (Table 1) were 204 subjected to PCR using primer set OTT_RS06375-F1/R615 (Table 2), which targets the full-length 205 ompA gene (OTT_RS06375) of annotated O. tsutsugamushi Ikeda. [65]. Amplicons of the expected size 206 were generated for all O. tsutsugamushi isolates except for SV400 and UT125. A second primer set 207 specific for OTT_RS06375 nucleotides 64 to 615 successfully amplified the targeted ompA region from 208 SV400 and UT125. Amplicons generated using the OTT_RS06375-F1/R615 and OTT_RS06375- 209 F64/R615 primer sets were cloned and sequenced. The ompA nucleotide and translated amino acid 210 sequences were deposited in GenBank. The nucleotide and amino acid identities ranged from 93.6% 211 to 100.0% and 90.6% to 100.0%, respectively, (Table S1, Figure 1, and Figure 2). SV400 and UT125 212 were excluded from heat map analyses because only partial sequences had been obtained for them. 213 Aligning all OmpA amino acid sequences revealed that several segments thereof were 100% 214 conserved among the isolates (Figure 3). 215 216 3.2 Molecular modeling of OmpA 217 218 OmpA proteins of A. phagocytophilum and A. marginale, which are in the order Rickettsiales with 219 O. tsutsugamushi, contribute to their abilities to bind and invade mammalian host cells [41,44]. We 220 previously demonstrated that these two OmpA proteins’ tertiary structures are highly similar and 221 residues that are critical for adhesin function are conserved between them and are presented as part 222 of surface exposed alpha helices [41,44,45]. To predict the tertiary structure of O. tsutsugamushi OmpA, 223 molecular modeling of Ikeda OmpA residues 21 to 204 (minus the signal sequence) as a 224 representative naturally occurring OmpA sequence was performed using the PHYRE2 recognition 225 server (www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi) [69], which generates three-dimensional models 226 for protein sequences and threads them on known crystal structures. The resulting model is 227 presented in Figure 4A. The O. tsutsugamushi OmpA tertiary structure was evaluated for similarity 228 to Anaplasma spp. OmpA. Because the A. phagocytophilum and A. marginale OmpA models are nearly 229 identical [41], the O. tsutsugamushi OmpA predicted structure was compared only to that of A. 230 phagocytophilum. Threading the two models onto each other using PyMOL (pmyol.org/educational) 231 revealed their folded portions to be very similar structurally with the exception of O. tsutsugamushi 232 residues 21 through 50, which are disordered (Figure 4B). Notably, O. tsutsugamushi OmpA bears a 233 surface-exposed alpha helix that overlays with the functionally essential surface-exposed alpha helix 234 of A. phagocytophilum OmpA. Moreover, aligning the A. phagocytophilum (L59KGPGKKVILELVEQL74) 235 and A. marginale OmpA binding domains (I53KGSGKKVLLGLVERM68) with the analogous region of 236 O. tsutsugamushi OmpA (L103SEESKRVLRAQSAWL118) indicated conservation of several residues 237 including a lysine that is critical for A. phagocytophilum OmpA and A. marginale OmpA adhesin 238 function [41,45] (Figure 4C). This region is identical among all O. tsutsugamushi translated OmpA 239 sequences in this study with the exception of residue 116 (Figure 3 and Figure 4). Thus, O. 240 tsutsugamushi OmpA is predicted to exhibit high structural similarity to OmpA proteins of other

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Color Key/Histogram 0 0 4 0 0 3 t n 0 u 0 o 2 C 0 0 1 0 1 2 3 4 6 7 8 9 6 0 . 3 0 7 4 1 8 5 2 0 3 ...... 1 9 4 5 5 6 7 7 8 9 9 9 9 9 9 9 9 9 Value UT177 FPW2016 UT418 Volner UT559 UT76 MAK110 SV484 LNT1301 MAK243 UT340 TH1817 SV445 AFPL12 AFC30 TM2532 TM2328 TA763 UT336 TH1812 Karp 18−032460 Kato Ikeda CRF136 AFC3 CRF09 Calcutta UT169 UT652 MAK119 LNT1189 LNT1153 LNT1310 TM2494 TM2766 CRF58 CRF158 TM2950 Gilliam TM2395 FPW2049 FPW1038 UT219 TM3115 Citrano Woods Brown Boryong r s g n o 5 9 8 9 5 0 8 8 6 4 0 3 9 9 2 9 a 9 3 6 a o 0 p 2 6 3 8 2 0 2 5 7 0 3 1 4 0 6 9 8 6 7 m t t e r d n n 1 1 3 4 9 5 5 5 6 9 1 5 8 1 5 6 t 0 3 d 6 1 3 6 2 3 3 1 4 1 4 4 0 8 1 7 5 1 1 7 w C a a n a o i o a 1 2 0 0 3 9 1 7 4 3 1 1 1 6 1 u 1 e 4 8 3 7 3 5 L 4 8 3 2 3 4 1 5 l 4 0 1 F F T o l C F r l K y r o c k K 3 1 2 2 2 2 2 1 1 1 2 1 2 2 1 1 o 2 t T i F T T F T T T T T K A P V K V K l I R R F U i r A 3 B T T T T T V a H F H U R A U U R U S U A S A U U U o M M G M M M M M W A W W C C W C 0 N N N T T N . A T T T T T C T T C C B P P M M M P L L L L 8 F F F 1 X 241 242 243 Figure 1. Heat map of percent nucleotide identity of the ompA DNA sequences among 49 O. tsutsugamushi 244 isolates. The heat maps were generated using the pairwise identity matrix tables with hierarchical clustering 245 method. The names of the isolates are provided on the right side and bottom of the heat map. Dendrograms are 246 on the left side and on top of the heat map. 247

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Color Key/Histogram 0 0 6 0 0 5 0 0 4 t n 0 u 0 o 3 C 0 0 2 0 0 1 0 4 9 3 8 2 7 1 6 6 0 . 6 6 7 7 8 8 9 9 0 0 ...... 1 9 1 2 3 4 5 6 7 8 9 9 9 9 9 9 9 9 Value SV445 FPW2016 UT177 UT340 UT418 TH1817 AFC30 AFPL12 MAK243 Calcutta Volner TM2532 TH1812 MAK110 UT169 UT76 TM2328 UT336 UT559 Karp LNT1301 TA763 SV484 18−032460 UT652 MAK119 Kato CRF136 CRF09 Ikeda AFC3 Gilliam TM2395 FPW2049 UT219 FPW1038 Boryong Citrano Brown Woods LNT1189 LNT1153 LNT1310 TM2494 TM2766 CRF58 TM2950 TM3115 CRF158 r s 8 5 0 6 4 0 9 8 3 n o g 8 9 9 5 a 9 3 6 o 9 2 0 4 3 1 p 9 6 8 6 9 0 2 2 a 3 2 0 7 8 0 7 6 5 m t t e r d 5 1 5 6 9 1 8 5 5 n n 3 1 4 9 d 0 3 1 5 6 8 6 0 5 3 2 7 6 1 1 3 t 4 1 3 1 1 4 7 1 4 w C a a n a o i 1 1 9 7 4 3 1 1 a o 0 2 0 3 e 1 1 6 4 4 7 3 5 3 3 1 1 8 5 l u 2 L 8 4 3 1 0 4 F F T o l C F r l K o r y k K c 3 2 2 2 1 1 1 1 2 2 2 1 2 1 2 o 1 2 t F T i F T T T T T T T K V A K K P V l I R R U F i r A 3 T T T B T T V H a F H R U R A U S U U U A A U U U S M M M M o M G M M W A C W W C W C 0 N N N N T T . A T T T T T T T C C C B P P M M M P L L L L 8 F F F 1 X 248 249 Figure 2. Heat map of percent identity of the OmpA translated amino acid sequences among 49 O. tsutsugamushi 250 isolates. The heat maps were generated using the pairwise identity matrix tables with hierarchical clustering 251 method. The names of the isolates are provided on the right side and bottom of the heat map. Dendrograms are 252 on the left side and on top of the heat map. 253

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254 255 Figure 3. Alignment of translated O. tsutsugamushi isolate OmpA sequences. Presented is a ClustalW alignment 256 of the translated amino acid sequences of all 51 O. tsutsugamushi isolates in this study. Amino acid differences 257 relative to the majority (consensus) sequence are denoted by black shaded white text. Sequence gaps relative to 258 the majority are indicated by dashes. Residues 1-21 for SV400 and UT125 could not be predicted because ompA 259 could be amplified by OTT_RS06375-F64/R615, but not OTT_RS06375-F1/R615.

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260 Rickettsiales members that are important for infection, its surface-exposed alpha helix is identical 261 among all isolates examined herein, and residues of the alpha helix are identical to those of Anaplasma 262 spp. OmpA proteins that are key for pathogenicity. 263 A. B.

C. Ot 103-LSEESKRVLRAQSTWL-118 Ap 59-LKGPGKKVILELVEQL-74 Am 53-IKGSGKKVLLGLVERM-68 264 265 Figure 4. Molecular modeling of O. tsutsugamushi OmpA reveals structural similarity with A. phagocytophilum 266 OmpA and conservation of residues that are critical for Anaplasma spp. OmpA function. (A) Predicted tertiary 267 structure for O. tsutsugamushi OmpA. The OmpA mature protein sequence was predicted using the PHYRE2 268 algorithm. The cyan portion corresponds to residues 103 to 118, which are analogous to residues 59 to 74 and 53 269 to 68 of A. phagocytophilum and A. marginale OmpA predicted tertiary structures, respectively, that form receptor 270 binding domains. Magenta residues are those that vary among the 51 O. tsutsugamushi OmpA sequences studied 271 herein. (B) Overlay of O. tsutsugamushi and A. phagocytophilum predicted OmpA tertiary structures. A. 272 phagocytophilum OmpA residues are labeled dark blue except for residues 59 to 74 (labeled orange) that lie within 273 a surface exposed alpha helix and constitute the receptor binding domain. O. tsutsugamushi OmpA residues are 274 labeled gray except for residues 103 to 118 (labeled cyan) that are analogous to A. phagocytophilum OmpA 275 residues 59 to 74. (C) Alignment of O. tsutsugamushi (Ot) OmpA residues 103 to 118, A. phagocytophilum (Ap) 276 amino acids 59 to 74, and A. marginale (Am) residues 53 to 68. Identical residues among the three sequences are 277 shaded gray. Similar amino acids are underlined. Functionally essential residues in the Anaplasma spp. OmpA 278 proteins are in red text. 279 280 3.3 Development of a primer set that specifically amplifies an ompA sequence unique to O. tsutsugamushi 281 282 Next, it was examined if a primer set could be devised that would amplify a segment of O. 283 tsutsugamushi ompA from all O. tsutsugamushi isolates in this study. A BLASTN search against 284 GenBank using the O. tsutsugamushi Ikeda OTT_RS06375 sequence as query determined that 285 nucleotides 64 to 279 were unique to O. tsutsugamushi isolates. Examination of nucleotides flanking 286 and within this region for sequences that would have annealing temperatures compatible with 287 thermal cycling conditions denoted nucleotides 57 to 85 (primer ompA-57F; Table 2) and 232 to 260 288 (primer ompA-260R; Table 2) as potential primers. For nucleotides 57 to 85, 32 of the 51 isolates had a 289 sequence that was identical to the consensus, 17 had a single nucleotide mismatch, and one (Boryong) 290 had three nucleotide mismatches (Figure 5A). For isolates SV400 and UT125, which ompA sequences 291 were identified using primer set OTT_RS06375-64F/615R and therefore began with nucleotide 64, 292 identity of nucleotides 57 through 63 could not be determined. However, nucleotides 64 through 85 293 for these two isolates exactly matched the consensus. All isolates exhibited 100% identity among 294 nucleotides 232 to 260 except for two (FPW1038 and UT219), each of which had a single nucleotide 295 mismatch (Figure 5B). For all isolate target sequences of the ompA-64F/260R primer set that had 296 nucleotide mismatches, only one (UT336) exhibited a nucleotide mismatch near the 3’ end of either 297 primer. Importantly, the UT336 nucleotide mismatch for ompA-64F did not occur within the final two 298 nucleotides. Thus, it was expected that the primer set would amplify ompA from all 51 isolates. 299

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A.

57 60 70 80 Ikeda GTGGAAATGTTTATGGC AAAGATCTAAAC FPW1038 ------C------SV484 ------C------UT652 ------C------Boryong ------C---G-A----- CRF58 ------G------CRF158 ------G------FPW2049 ------G------LNT1153 ------G------LNT1189 ------G------LNT1310 ------G------TM2395 ------G------TM2494 ------G------TM2766 ------G------TM2950 ------G------TM3115 ------G------MAK119 ------A------Citrano ------C------UT336 ------G--

B.

232 240 250 260 Ikeda GATGTAAAGCATGA ACAGTTTTTACAAGC FPW1038 ------G------UT219 ------G------300 301 Figure 5. Nucleotide mismatches in the binding sites for ompA-64F and ompA-260R among O. tsutsugamushi 302 isolates examined in this study. The ompA-64F (A) and ompA-260R (B) sequences, both of which correspond to 303 O. tsutsugamushi Ikeda nucleotides, are listed. Below each are the corresponding nucleotides of where each 304 primer would bind for any isolate in this study that has at least one mismatch. Numbers above the sequences 305 refer to the nucleotide positions in Ikeda ompA. Dashes indicate identical nucleotides to those of ompA-64F and 306 ompA-260R. Mismatches in the primer sequences are denoted by black shaded white text with the specific 307 nucleotide difference indicated per sequence below. 308 309 To confirm the efficacy of the ompA-64F/260R primer set, it was evaluated for the ability to 310 amplify its target from six representative isolates that had one or more nucleotide mismatches at the 311 primer binding sites (Boryong, LNT1153, SV484, UT336, MAK119, Citrano) plus SV400 and UT125, 312 for which it was unknown whether ompA-64F would bind. A band of the expected size was generated 313 for all eight samples, but not for negative control reaction that lacked DNA template (Figure 6). Next, 314 the primer set was examined whether it would non-specifically generate products from multiple 315 eubacterial human pathogens and Rickettsia spp. The primer set failed to yield PCR products from 316 the 20 samples examined but produced an amplicon of the expected size from O. tsutsugamushi Ikeda. 317 (Figure 7A), which has a sequence that is identical to the consensus binding sites for both primers. 318 PCR products could be generated from the eubacterial and Rickettsia spp. using primers targeting 319 eubacterial 16S rRNA and the rickettsial gene encoding 17-kDa protein [36] (Figure 7, B and C), 320 thereby confirming sample integrity. Overall, these data demonstrate that the ompA-64F/260R 321 amplifies DNA target sequences even if they contain limited nucleotide mismatches. 322

3 g 5 9 n 1 4 6 1 o 0 5 l o 1 8 3 1 n 0 2 tr y a r T 4 3 K r 4 1 c A t ) o N V T i V T - B L S U M C S U ( 300 – 200 – 323 324 Figure 6. Primer set ompA-64F/260R amplifies ompA sequences having one to three nucleotide mismatches in the 325 primer binding sites. DNA isolated from tissue culture cells infected with each of the indicated O. tsutsugamushi 326 isolates or no template control [(-) ctrl] were subjected to PCR analysis using ompA-64F/260R primers. The 327 numbers to the left of the panel correspond to DNA ladder sizes. Data are representative of three experiments 328 with similar results. 329

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A. 0 0 5 B. $2 K F la S 2 K a X i $ 1 X il O h th 6 h # p u #O s i s e R p u li o i t is o ii s i n i e a e V il t a b m i ic a i m n ii e c u o c n c r b o n i ra li e s ti n rs i a a i u s ic s i n s fr k ir l e t n r l o n i i e e o n in s e e tr m c p v r s p a a ri s # # # # # # # # # #m #c p v # # p $c ...... # .# . . .# ) 400 P E L B N N F R R P E L B N N F (@ – 500 – – – 300$– 400$– – – 200 300 a d T e R l k IT $ n n I $ 1 i o i 7 9 re t h C. S 9 s 8 g s i i $ B n H 5 s l R 6 i i u 0 P n a 4 i 0 R V k m m 5 $R l s V e C h i 2 il a n n i p i e 2 $ 1 i l ii n ri s a z g $ e to a r a e tt a W F 7 9 r r t e c ic i u S 9 s 8 g t o n k i e r w s S 2 i i $ B ii s n r p i h t $ H 5 s l R 6 i in o i k b o p u l 1 i u o a c i r tr th 6 P R n a V 4 m m c m p h i y s h k l #a # # # #r #r #s #p #t #t c e R s V e C ii 2 e i m ...... )$ li i# n i p z @ e V i r e ts a W o R R R R R R R R R O ( i ra r ta e t c a i ly 400 a r t o ic e i – ic n k ir w h b r a s n o r ip k o p l – f k u o a c ib r m tr 300$– a a a c m p rh ri s p ty a # # # $ # # # # # # # # $c –– ...... ) 200 R R R R R R R R R R R R (@ 200$– 330 100$– 331 Figure 7. Confirmation of ompA-64F/260R specificity. DNA samples from eubacterial and Rickettsia spp. were 332 subjected to PCR using (A) ompA-64F/260R. The eubacterial and Rickettsia spp. DNA samples were also subjected 333 to PCR using primer sets targeting eubacterial 16S rRNA (B) and the rickettsial gene encoding the 17-kDa protein 334 (C) to verify sample integrity. For the experiment in (A), O. tsutsugamushi Ikeda DNA was included as a positive 335 control. A no template control [(-) ctrl] was included in each experiment. Vertical lines between lanes indicate 336 that irrelevant lanes from the gel images were removed. Data are representative of two experiments with similar 337 results. 338 339 3.4 Evaluation of the O. tsutsugamushi ompA-specific primer set in qPCR 340 341 To determine the detection limit of ompA-64F/260R, the primer set was evaluated by qPCR using 342 as template plasmid pCR2.1-ompA, which has ompA nucleotides 1 to 615 inserted, serially-diluted 343 from 1 x 106 to 1 x 10-2 copies per reaction. The primers detected ompA as low as 101 copies (R2 = 0.995) 344 (Figure 8A). The experiment was repeated with reactions containing pCR2.1-ompA diluted from 100 345 to 3.5 copies. The primers reliably detected ompA DNA at a concentration as low as 3.5 copies (R2 = 346 0.950) (Figure 8B). Thus, ompA-64F/215R has an approximate detection limit of 3.5 copies. 347 A. B.

37 35 36 35 q

q 30

C 34 C 25 33 32 20 31 1.0 2.0 3.0 4.0 5.0 6.0 0.5 1.0 1.5 2.0 Log-starting-quantity Log-starting-quantity 348 349 Figure 8. Primer set specific for O. tsutsugamushi ompA is capable of amplifying low copy number ompA DNA 350 standards. Standard curves generated by ompA-64F/260R amplification of ompA DNA standards diluted ten- 351 fold from 1 x 106 to 1 x 10-2 (A) and from 100 to 3.5 copies (B). Data are representative of two experiments with 352 similar results. 353 354 Next, the ompA-64F/260R primer set was evaluated using qPCR for the ability to detect and 355 quantify ompA copies in DNA samples isolated from organs harvested from Swiss CD-1 mice infected 356 with O. tsutsugamushi Gilliam. The CD-1 mouse intradermal inoculation model was recently 357 demonstrated to exhibit features of early scrub typhus infection in humans, including distant organ 358 dissemination. Gilliam was one such strain that was evaluated using this model [68]. Reactions 359 performed on the same plate with pCR2.1-ompA diluted ten-fold from 1 x 106 to 1 x 100 copies allowed 360 for copy number quantitation. Negative control reactions consisted of those containing DNA isolated 361 from organs of mock inoculated mice and those lacking DNA template. The primers detected ompA 362 at copy numbers of 71.0 + 5.2, 39.3 + 22.2, 184.0 + 22.2, and 181.0 + 49.8 in kidney, liver, lung, and

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363 spleen DNA samples, respectively, recovered on day 6 from a mouse infected with O. tsutsugamushi 364 Gilliam, but failed to amplify ompA from DNA isolated from heart of the infected mouse or from a 365 mock inoculated mouse (Figure 9A). When qPCR was performed on DNA samples isolated from 366 organs of O. tsutsugamushi Karp infected mice on days 10, 14, and 21, ompA DNA was detected in all 367 samples and at the highest levels for each organ in samples isolated on day 14 (Figure 9B). Copy 368 numbers of ompA for these samples ranged from 22.4 + 4.98 (heart 10 d post-infection) to 1,890 + 219.0 369 (lung 14 d post-infection). Lungs having the highest level of ompA DNA is consistent with the 370 bacterial burden being the greatest in the lungs in this mouse model [68]. These data demonstrate the 371 ability of ompA-64/215 to amplify and quantify ompA copies in DNA samples recovered from tissues 372 of O. tsutsugamushi infected mice. 373 A. B. A 250 A 3000 N Heart N D D % % g Kidney g 2500 n n

% 200 % 0 0

5 Liver 5 / / 2000 r r e 150 Lung e b b m Spleen m 1500 u u n n % 100 % y y 1000 p p o o c 50 c A

A 500 p p m m o 0 o 0 Mock%inoculated Infected 10%dpi 14%dpi 21%dpi 374 375 Figure 9. The ompA-64F/260R primer set amplifies ompA in qPCR from DNA recovered from the organs of O. 376 tsutsugamushi infected mice. Fifty ng of DNA isolated from the indicated organs from Swiss CD-1 mice that had 377 been mock inoculated, infected with the O. tsutsugamushi Gilliam on day 6 post-infection (dpi; A), or from O. 378 tsutsugamushi Karp on 10, 14, or 21 dpi (B). Data are representative of at least three experiments with similar 379 results.

380 4. Discussion 381 Scrub typhus is a global health concern for which neither a vaccine that provides homologous 382 protection nor a reliable diagnostic assay exists. To effectively protect against or detect the diversity 383 of O. tsutsugamushi strains, the bacterial target must be highly conserved. OmpA satisfies this 384 criterion, as it displays 93.6% to 100.0% and 90.6% to 100.0%, conservation at the nucleotide and 385 protein levels, respectively, among the isolates in this study that originated from multiple Asia- 386 Pacific locations. 387 388 While the role of OmpA in O. tsutsugamushi pathobiology is unclear, studies of other Rickettsiales 389 members’ OmpA proteins offer precedents that O. tsutsugamushi OmpA likely contributes to and 390 could be immunologically targeted to inhibit infection. OmpA proteins of A. phagocytophilum, A. 391 marginale, E. chaffeensis, and R. conorii are each on the bacterial surface, participate in host cell entry, 392 and can be targeted by antibodies to inhibit infection in vitro [38,41,42,44,45]. Patients naturally 393 infected with A. phagocytophilum or R. conorii and animals experimentally infected with A. 394 phagocytophilum or E. chaffeensis develop antibodies that recognize recombinant forms of the 395 respective OmpA proteins in serological assays [38,44,46], indicating that Rickettsiales bacteria express 396 OmpA during in vivo infection. The abilities of A. phagocytophilum and A. marginale OmpA to mediate 397 bacterial adhesion to and invasion of host cells relies on receptor binding domains that consist of 398 specific lysine and glycine residues within the proteins’ structurally conserved, surface-exposed 399 alpha helices. Antisera specific for these binding domains inhibits Anaplasma spp. infection of host 400 cells [41,45]. Strikingly, the predicted O. tsutsugamushi OmpA tertiary structure is very similar to that 401 of A. phagocytophilum OmpA, so much so that their aforementioned alpha-helices and a lysine residue 402 thereof overlay when the proteins are superimposed on each other. It will be important to confirm

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403 whether O. tsutsugamushi OmpA is surface-exposed and if antiserum raised against the full-length 404 protein or its alpha helix amino acids 103-118 inhibits infection of host cells. 405 406 A R. prowazekii ompA deletion mutant retains the ability to productively infect mice [70], likely 407 due to OmpA being one of many OMPs that cooperatively function to mediate invasion of host cells 408 [71-73]. Nonetheless, it is worth investigating whether immunization against OmpA offers protection 409 from O. tsutsugamushi challenge. A humoral immune response against OmpA or a key portion thereof 410 together with other conserved OMPs could inhibit bacterial entry into host cells, which would 411 essentially lead to its demise due to its obligatory intracellular lifestyle. This very concept has been 412 demonstrated for blocking A. phagocytophilum infection in vitro: whereas OmpA binding domain 413 antibody alone reduces infection by approximately 25%, an antibody cocktail targeting the binding 414 domains of OmpA together with two additional OMPs nearly eliminates infection of host cells [44,45]. 415 Anti-OmpA antibodies could also eradicate O. tsutsugamushi load in vivo via complement-mediated 416 killing or opsonophagocytosis. Indeed, although the exact mechanism is unclear, guinea pigs 417 immunized with a recombinant form of truncated R. heilongjiangensis OmpA exhibited reduced 418 bacterial load, organ pathology, and interstitial following challenge with R. 419 heilongjiangensis or R. rickettsii compared to sham-immunized animals [49]. 420 421 Exploiting the high degree of ompA nucleotide conservation facilitated development of primer 422 set OTT_RS06375-64F/615R, which amplified its target from all O. tsutsugamushi isolates examined 423 herein including ompA sequences having up to three nucleotide mismatches in the primer binding 424 sites. In qPCR, OTT_RS06375-64F/615R detected ompA at a copy number as low as 3.5. This sensitivity 425 level rivaled or exceeded that reported for other qPCR assays [34,74,75]. Moreover, these primers 426 detected ompA in DNA isolated from organs of O. tsutsugamushi infected mice. The ability of 427 OTT_RS06375-64F/615R to detect ompA at a low copy number and to do so in the presence of excess 428 host tissue-derived DNA evidence its potential for sensitively detecting O. tsutsugamushi ompA in 429 DNA isolated from scrub typhus patient-derived samples such as eschar swabs, blood, or buffy coats, 430 as has been demonstrated for other qPCR assays [34,74,76-81].

431 5. Conclusions 432 The high degree of nucleotide and amino acid conservation of OmpA among diverse O. 433 tsutsugamushi isolates and its structural similarity to other Rickettsiales OmpA proteins that have been 434 successfully targeted to inhibit bacterial invasion of host cells argue for its consideration as a vaccine 435 candidate that could provide homologous protection and a molecular target that could be useful in 436 diagnosing scrub typhus infections.

437 Supplementary Materials: The following are available online at www.mdpi.com/link, Table S1: Nucleotide and 438 amino acid identity values for O. tsutsugamushi OmpA sequences. 439 Acknowledgments: This study was supported by the National Institutes of Health (grants AI123346 and 440 AI128152 to Jason A. Carlyon), American Heart Association (grant 13GRNT16810009 to Jason A. Carlyon and 441 grant 13PRE16840032 to Lauren VieBrock), the U.S. Military Infectious Diseases Research Program (grant A1230 442 to Allen L. Richards), and Virginia Commonwealth University (Presidential Research Quest Fund grant to Jason 443 A. Carlyon). The views expressed in this presentation are those of the authors and do not necessarily represent 444 the official policy or position of the Department of the Navy, Department of Defense, U.S. Government. 445 Author Contributions: J.A.C., S.M.E., L.V., A.L.R., and R.T.M. conceived and designed the experiments and 446 analyses; S.M.E., H.E.A., L.V., A.L.F., and S.C. performed the experiments; J.A.C., S.M.E., L.V., H.E.A., R.S.G., 447 and J.A.C. analyzed the data; D.P., A.L.R., and J.J. contributed reagents and materials; J.A.C. wrote the paper. 448 Conflicts of Interest: The authors declare no conflict of interest. The funding sponsors had no role in the design 449 of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the 450 decision to publish the results. 451

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